Inhibition of BK channel activity by association with calcineurin in rat brain

Large conductance calcium-activated potassium (BK(Ca)) channels are regulated by a number of different protein kinases and phosphatases. The close association of enzymes and channel have been shown to underlie many examples of modulation. However, only the association of protein kinase A with the BK(Ca) channel has been detailed [Tian et al. (2003)J. Biol. Chem., 278, 8669-8677]. We have found using reciprocal immunoprecipitations that the BK(Ca) channel associates with the calcium/calmodulin-dependent phosphatase calcineurin, in Wistar rat brain. A HA-tagged construct of the carboxyl terminus of rSlo(27), a variant of the BK(Ca) channel that is abundant in the hippocampus [Ha et al. (2000)Eur. J. Biochem., 267, 910-9218], was found to associate only with the B subunit of calcineurin. This data suggests that the majority of the interaction of the BK(Ca) channel with calcineurin is mediated by the B subunit of the phosphatase. This was confirmed by using glutathione-S-transferase (GST) fusion proteins of the linker regions between the S7-S10 hydrophobic domains in the carboxyl terminus of rSlo(27), where only the B subunit of calcineurin interacted with regions between S7 and S9 of the channel. Addition of a constitutively active calcineurin (CaN(420)) to inside-out membrane patches excised from cultured hippocampal neurons resulted in a dramatic reduction in BK(Ca) channel open probability, with only very short-duration events being apparent. These data suggest that BK(Ca) channel activity is inhibited by calcineurin, an effect mediated by the association of the calcineurin B subunit with the carboxyl terminus of the channel.